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- Materials and Methods
In Western Europe, heathlands and matgrass swards were once common vegetation types (De Smidt, 1975; Gimingham, 1992). From the mid-eighteenth century, however, European heathlands declined dramatically in area, mainly as a result of reclamation for forestry and farming (De Smidt, 1975, 1979; Webb, 2002). Their unique composition of many characteristic and endangered plant and animal species makes these ecosystems subject to conservation and restoration (Gimingham, 1992; Webb, 1998).
In addition to losses through habitat destruction, the biodiversity of dry and wet heathlands and related matgrass swards is rapidly declining (Roelofs et al., 1996; Bobbink et al., 1998). Plant species diversity in heathlands in the Netherlands has declined over 50% in the past 50 yr because of environmental stresses (Vonk et al., 2001), and many herbaceous species such as Antennaria dioica, Arnica montana, Cirsium dissectum, and Gentiana pneumonanthe have largely disappeared and are seriously threatened. Conversely, grasses such as Deschampsia flexuosa and Molinia caerulea have become dominant.
It was suggested that this shift towards grass-dominated vegetation was caused by the increased atmospheric deposition of nitrogen- and sulphur-containing compounds during the past decades, resulting in eutrophication and acidification of the soil (Heil & Diemont, 1983; Bobbink et al., 1998). Earlier studies (Heil & Diemont, 1983; Berendse & Aerts, 1984; Roelofs, 1986; Aerts et al., 1990) showed that grasses had higher productivity at elevated N concentrations compared with herbaceous species and shrubs. Grasses were therefore thought to outcompete herbaceous species and shrubs at high N availability (Aerts & Berendse, 1988; Pitcairn et al., 1991). However, a study by Houdijk et al. (1993) showed that many herbaceous species had already disappeared from the heathlands before grasses became dominant, indicating that processes other than competition for light and nutrients play an important role in the decline of herbaceous species.
Nitrogen deposition in the Netherlands is mainly in the form of ammonium (NH4+) (Erisman, 1990; Bobbink et al., 1992; Boxman et al., 1998; Kreutzer et al., 1998) and, as a result, plants encounter increased NH4+ concentrations which have been shown to be toxic for many plant species (Mehrer & Mohr, 1989; Britto & Kronzucker, 2002 and references therein), including herbaceous heathland species such as A. montana and C. dissectum (De Graaf et al., 1998; Dorland et al., 2003; Lucassen et al., 2003).
In addition, the atmospheric deposition of S and N results in acidification of heathland soils and the depletion of buffering base cations such as Ca2+, Mg2+ and K+ (Carnol et al., 1997). This may lead to a change in soil buffering and the loss of acid-sensitive species (Houdijk et al., 1993). For degraded, acidified, dry heathlands an average soil pH of 3.8–4.2 was found, whereas for degraded, acidified, wet heaths a slightly higher pH of 4.0–4.5 (caused by reduction processes) was measured (De Graaf et al., 1994; Roelofs et al., 1996; Dorland et al., 2003). Acidification in wet heaths is also induced by lowering of groundwater tables, as this enhances acidifying processes such as the oxidation of iron sulphides, mineralization and the influence of acidic rainwater (Roelofs, 1993; Grootjans et al., 1996; Runhaar et al., 1996; Lamers et al., 1998). Because of the acid soil conditions, nitrification in heathland soils is low and is strongly reduced with decreasing pH (Roelofs et al., 1985; Van Breemen & Van Dijk, 1988; Dorland et al., 2004). As nitrification is an acidifying process, the inhibition of nitrification constitutes a negative feedback on acidification. All these processes contribute directly or indirectly to an accumulation of NH4+ in the soil, mostly accompanied by higher NH4+ availabilities.
Although external pH was suggested as the primary cause of the decline of herbaceous species in favour of grasses (Van Dam et al., 1986; Dueck & Elderson, 1992; Houdijk et al., 1993), others found no effects of external pH on the growth of herbaceous heathland species (Van Dobben, 1991), or found indirect pH effects through aluminium toxicity (Heijne et al., 1996). NH4+ concentrations did not explain the dramatic decline in plant diversity either, as many herbaceous species were shown to grow well on high NH4+ concentrations under weakly buffered conditions (Bobbink et al., 2003).
The relationship between NH4+ toxicity and soil acidification has been the subject of a number of studies (Findenegg, 1987; Dijk & Eck, 1995; Dijk & Grootjans, 1998; Lucassen et al., 2003). Lucassen et al. (2003) suggested that the decline of C. dissectum was caused by the combination of high NH4+ concentrations and a low pH of the growth medium. They hypothesized that at high NH4+ concentrations and low external pH, C. dissectum suffered from low internal pH levels as a result of reduced proton excretion. This is explained by proton excretion which was found to occur when NH4+ is assimilated in plants (Raven & Smith, 1976; Findenegg, 1987; Van Beusichem et al., 1988; Goodchild & Givan, 1990). Others also found that growth on NH4+ results in a decrease in tissue pH (Gerendás et al., 1990; De Graaf et al., 2000).
In this study we describe the results of a hydroponic experiment with five heathland species from both wet and dry heaths: G. pneumonanthe, Succisa pratensis, Calluna vulgaris, A. dioica and D. flexuosa. Plants were subjected to environmental stress by growing them in media with different NH4+ concentrations and pH levels. Biomass, mortality, internal pH of plants and the chemical composition of plants were measured to estimate the fitness and survival of plants. NH4+-tolerant species are usually found in acidic habitats and are likely to be adapted to NH4+ nutrition, as NH4+ is the dominant N form at low pH (Gigon & Rorison, 1972; Troelstra et al., 1990). In contrast, species from less acidic habitats usually prefer nitrate (Falkengren-Grerup & Lakkenborg-Kristensen, 1994; Britto & Kronzucker, 2002). Therefore we hypothesized that acidification would negatively affect the survival and fitness of A. dioica and S. pratensis as these are characteristic for weakly buffered conditions and are regarded as acid-sensitive species. It was also expected that these effects will be enhanced by increased NH4+ concentrations. Calluna vulgaris and D. flexuosa can be found in eutrophied and acidified heathlands and are therefore thought to be more acid-tolerant. These species were expected to be less affected by low external pH in combination with high NH4+ concentrations. The rare herbaceous species G. pneumonanthe is regarded as slightly acid-tolerant and thus less susceptible to low external pH than A. dioica and S. pratensis.